Skin cosmesis treatment with clostridia neurotoxins

ABSTRACT

Methods of using clostridial toxins and other biological agents to treat skin cosmesis in humans is provided. The disclosed methods provide beneficial effects in humans.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/870,761 filed Aug. 27, 2010; which is a continuation in part of U.S.patent application Ser. No. 11/904,963 filed Sep. 28, 2007, now U.S.Pat. No. 7,824,693; which is a continuation in part of U.S. patentapplication Ser. No. 10/524,304 filed Feb. 8, 2005, now U.S. Pat. No.7,288,259; which is a 371 application of PCT/US03/25708 filed Aug. 18,2003; which claims priority to U.S. Provisional Application Ser. No.60/404,378 filed Aug. 19, 2002, all of which are incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for thinning the skinand treating fine wrinkling of skin, by administration of clostridianeurotoxins in particular Botulinum Toxin. This invention also relatesto methods and compositions for treating of skin, particularlyundesirable secretions by holocrine glands by administration ofclostridia neurotoxins in particular Botulinum Toxin.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to asreferences, within parentheses or by footnote. The procedures set forthin these publications where relevant are hereby incorporated byreference. The reasoning set therein is also incorporated by referencein so far as it does not differ from or conflict with the text herein.In case of such difference or conflict the text herein controls.

Cells contain vesicles (also called endosomes) that are sphericalstructures with a bilipid membrane. These endosomes can merge with thecell membrane and release their content into the extracellularenvironment (exocytosis). The process of forming vesicles and mergingthem with cellular membranes can be broadly divided into two categories:constitutive and regulated. Constitutive exocytoses are maintenancefunctions of the cell while regulated exocytoses is a specializedresponse of the cell to an external or internal signal. The paradigm ofspecialized regulated secretion is the release of neurotransmitters atneuronal synapses. At the proper signal (usually a drop in cell voltage)hundreds of vesicles merge with the cell membrane to release theirneurotransmitters. The neurotransmitters diffuse across the synapticspace to bind to and excite the postsynaptic membrane of a secondneuron.

Exocytosis requires specialized proteins on the vesicle and presynapticmembrane that are collectively known as the SNARE proteins. Removal ofany of these proteins can stop vesicle docking to membrane and block ordecrease neural signaling. One protein on the vesicle membrane calledVAMP (vesicle associated membrane protein) and one on the presynapticmembrane called SNAP (synapse associated protein) are the targets of thebotulinum and tetanus neurotoxins from the Clostridial bacterium.

Botulinum toxin (BT) is a potent neurotoxin produced by the anaerobicgram-positive bacterium Clostridia botulinum and the closely relatedspecies Clostridia butyricum and beratti. When spores of the Clostridiabotulinum are ingested they germinate and secrete BT that passes fromthe GI tract into the systemic circulation. The systemic spread of BTcauses the disease botulism that is characterized by widespreadneuromuscular paralysis.

BT is a protein consisting of a light and heavy chain that togetherweigh approximately 150 kilodaltons. BT works by a three-stagemechanism, binding, translocation into the neuron and molecular action,each of which is performed by separate 50 kilodalton domains. Thebinding and translocation domains make up the heavy chain, while thecatalytic action is performed by the single domain of the light chain.

At present seven immunologically distinct serotypes of the BT are known,named A, B, C, D, E, F and G. The effect of BT is to inhibit the releaseof neurotransmitters and neuropeptides by neurons. Although all BTserotypes interfere with proteins that cause the exocytosis of synapticvesicles from cells they each interfere with different proteins, ordifferent parts of the same protein. In clinical use each serotypeappears to differ in its potency in blocking different classes ofneurons.

BT binds to specific molecules present on neuron presynaptic membrane.After binding it is internalized into the neuron by formation of anendosome. When the interior of the endosome becomes acidic, the lightchain translocates across the membrane and is released into thecytoplasm. After translocation across the cell membrane the CT lightchains cleave the proteins involved in synaptic vesicle docking andrelease that are collectively known as SNARE proteins. The targets ofthe CT are the following:

BT A & E cleave SNAP-25 (synapse associated protein)

BT C cleave SNAP-25 and syntaxin

BT B, D, F & G cleave VAMP (vesicle associated membrane protein)

The vesicles within neurons contain classical neurotransmitters(acetylcholine, epinephrine, norepinephrine, dopamine, serotonin,glutamate, GABA and others) and/or neuropeptides (substance P,neurokinin A, calcitonin gene related peptide (CGRP), neuropeptide Y,interleukins, growth factors and others). BT has been shown to blocksecretion of all these molecules.

THE CLINICAL EFFECTS OF BOTULINUM NEUROTOXIN Voluntary Motor Nerves

The first and still primary use of BT is to block motor nervecommunication with muscle fibers. BT is injected within the targetmuscle. The BT is then internalized into motor neurons where itdecreases or stops the release of the neurotransmitter acetylcholine(AChE), thereby causing paresis or paralysis of the muscle. Scottintroduced the concept of localized muscular injections of BT for thespecific condition of strabismus (squint, crossed eyes). Later BT wasfound to be particularly useful for movement disorders such as tics,spasms, contractures, cramps and tremors. More recently, the injectionof BT into facial muscles has been found to ameliorate skin wrinklingand lines related to aging. Another recent application of BT injectionsis to decrease the pain accompanying muscle tension in conditions suchas headache and temporomandibular joint syndrome.

Autonomic Motor Neurons

The autonomic nervous system is divided into a parasympathetic systemand a sympathetic system. The parasympathetic neurons use acetylcholineas their neurotransmitter and they can be blocked with BT. Thesympathetic nervous system uses noradrenaline as its neurotransmitterwith the single exception of sweating) and this neurotransmitter is notblocked by BT. Effector neurons of the parasympathetic system innervateand control the contraction of smooth muscles. Injections of BT havebeen used to decrease tone in the smooth muscles of the lower esophagealsphincter, esophagus, stomach wall, and pyloric sphincter, sphincter ofOdi, anal sphincter, and urinary bladder.

Autonomic Secretory Neurons

In addition to their innervation of smooth muscle, neurons of theautonomic system control or modulate a wide variety of other functionssuch as the secretion of various glands throughout the body. BTinjections have been used to decrease gastric secretions including acidproduction, nasal and other respiratory secretions, and tearing.

Neuropeptides

In addition to the neurotransmitters released at localized synapticsites, many autonomic and sensory nerves can release neuropeptides alongpart or all of the length of the axons. These peptides are mostnoticeable in skin as mediators of inflammation, allergic reactions andpain. For example injury in a small area of skin causes reflexvasodilation in surrounding areas. These reactions are neurally mediatedand depend on the release of neuropeptides. Although the neurogenicvasodilation of skin is blocked by BT, whether other phenomenon such aspain and swelling are blocked is still controversial.

Tetanus Toxin

Tetanus toxin (TT) is produced by the Clostridium tetani bacterium. WhenClostridium tetani spores infect wounds they germinate and produce TT.The TT is taken up by peripheral nerves near the wound and transportedretrograde to the central nervous system. It then spreads by diffusionand further neural transport. At low doses TT blocks release of theinhibitory neurotransmitters GABA and glycine causing increased activityin motor and autonomic nerves. Clinically the condition is calledtetanus and is characterized by severe muscular spasms and autonomicinstability. However, at higher doses TT blocks all neurotransmissionand clinically this appears as a flaccid paralysis.

TT also works by a two-stage mechanism that is similar to BT. However;the major difference is that after the peripheral neuron internalizesthe TT via endosomes the TT is not released into cytoplasm. Instead theendosomes are actively transported back to the cell body of the neuron.Here TT is again released into all synapses. At low doses the blockingis selective for inhibitory neurons. However, at higher doses TT blocksall neurons both inhibitory and excitatory, centrally and peripherally.

TT also differs from BT in that it is taken up by more classes ofneurons and at lower doses then BT. As described above the effect ofexcitation or inhibition of a given neuron by TT are dose related.Peripheral block of a neuron requires 10-1000 times the dose that causesexcitation of that same neuron. However, hybrid molecules of TT, such asthose that combine the heavy chain of BT with the light chain of TT,could be expected to have the same dose effects of as whole BT.

Skin Secretory Glands

Secretion is the combined result of production of secretion byspecialized cells within a skin gland, and the expulsion of thesecretion from the gland and ducts by contraction of surroundingmuscle-like myoepithelial cells. In some secretory glands, such as themammary gland, increased expulsion has a feed back effect in stimulatingfurther secretory production. In addition, the number and amount ofsecretory and myoepithelial cells can be modulated, with proportionalchanges in the amount of secretion produced. Finally the act ofsecretion is often accompanied by vascular dilation around the gland,which is believed to aid the gland by increased delivery of nutrients.

Skin secretory cells produce their secretion by 3 basic mechanisms.

Apocrine glands are the common sweat glands present throughout the skinsurface that produce profuse watery secretion. Apocrine glands have asimple organization; the gland is composed of a coiled duct in the dermis with an open end that discharges onto the skin surface. They producea watery secretion that evaporates and cools the skin thereby playing arole in thermoregulation. Discharge of the secretion from the lumen ofthe ductal portion of the apocrine sweat gland is assisted by the actionof myoepithelial cells which surround the secretory portion of thegland.

The cells lining the ducts of apocrine glands produce the secretion by amerocrine mechanism. This terminology is confusing as it would appearthat this sweat glands should be called merocrine glands. However, thesweat glands were named before the exact mechanism of their cellularsecretion was known, and their original names have persisted.

Excessive sweating, formally known as hyperhydrosis, is a commoncondition. Hyperhydrosis can occur in any part of the body but primarilyaffects the forehead, axilla, palms and feet. Sanders and Shaari (U.S.Pat. No. 5,766,605) Walker (U.S. 20020086036) disclose a method oftreating hyperhydrosis using needle and jet injections of BT.

Eccrine glands are commonly thought of as specialized sweat cells thatproduce a secretion with high protein content. Eccrine sweat glands arefound in the axilla, in the areolae of the breast and around the anus.They are larger than apocrine sweat glands and produce a viscoussecretion into hair follicles. The secretion released by apocrine sweatglands is odorless but the bacteria metabolize the secretion anddecompose its proteins, thereby causing a strong odor, which is usuallyexperienced as unpleasant.

W003026602A2: Medicine For Preventing And Treating Bromidrosis disclosesthe use of BT injections of BT for decreasing the odor of sweat.

Holocrine glands are fundamentally different from apocrine and eccrineglands. The secretion is primarily lipid rather than water. Moreover thelipid secretion is not secreted from cells; instead the cell, calledacebocyte, accumulates large amounts of the secretion and then dies,releasing the lipid material together with cellular remnants.

The vast majority of holocrine glands are sebaceous glands that producea lipid secretion called sebum. Sebaceous glands usually have severalacini that open into a short duct. The sebum producing cells are presentin the acini and in the wall of the duct. Most sebaceous glands arecalled pilosebaceous glands because they secrete into a duct thatnormally opens into the upper part of a hair follicle. However, incertain areas of the body such as the lips the ducts open directly ontothe skin's surface. A variety of other holocrine organs are present inthe skin of the eyelids: meibomium glands, and glands of Zeiss and Moll.

The control of holocrine glands has long been known to involve systemichormones, particularly the male sex hormones called androgens. Androgensincrease during puberty in both males and females. Supporting theconnection between hormones and sebaceous gland function is that sebumproduction increases after puberty and its peak incidence is from ages12 to 22. Increased sebum production is also related to pregnancy,pre-menstrual period and to birth control medication.

The role of classical neurotransmitters such in sebum production isunclear. Anticholinergics appear to have little effect on sebumproduction. However, pilocarpine, a cholinergic agonist, increases sebumproduction when iontophoresed across the skin (Yosipovitch et al, Br JDermatology, 1995: 561-4). Evidence suggests that increased sebumproduction in response to cholinergics may be due more to expulsion ofaccumulated sebum rather then increased cellular secretion. Even facialmovement seems to be important in emptying accumulations of sebum aspatients with facial paralysis accumulate greater amounts of sebum.

Dopamine appears to play an inhibitory role in sebum secretion aspatients with Parkinson's disease, a disease in which central nervoussystem levels of dopamine are low, have been reported to have increasedsebum production. Treatment of these patients with dopaminergic drugtherapy appears to decrease sebum levels whereas anticholinergic drugshave no effect (Villares, Arq Neuropsiquiatr, 1989, 47: 31-8). However,the dopaminergic effects on sebum production may be within the centralnervous system, or to increase facial movement as untreated Parkinson'spatients have decreased facial movement.

Recent research has shown that surprisingly, holocrine secretion iscontrolled by various neuropeptides, with substance P playing asignificant role (Toyoda and Marohashi, Med Electron Microsc 2001,29-40). Other neuropeptides found in neurons surrounding sebaceousglands include NPY, VIP and ENK, although their roles are unclear.

Mixed glands are skin secretory glands in which holocrine components aremixed with apocrine or eccrine components. Holocrine components havebeen reported in the cerumen glands that produce ear wax (Main and Lim,Laryngoscope, 1976, 86:1164-76) and mammary glands that produce milk.

CLINICAL CONDITIONS AFFECTING HOLOCRINE GLANDS Acne Vulgaris

One of the most common disorders of the sebaceous glands is AcneVulgaris (acne). Acne is largely a disease of adolescence and youngadulthood characterized by inflamed glands within the skin of the face,shoulders, and back. It is estimated that almost all people suffer atleast some acne during their lives.

Excessive sebum production within pilosebaceous glands results in anenlarged and obstructed sebum gland. These obstructed glands are highlysusceptible to infection by Propionibacterium acnes (P. acnes) causingan inflamed pustule called a comedone. These inflamed pilosebaceousglands can cause permanent scaring of skin.

Current therapy of acne includes topical and oral agents. Topicalretinoic acid is the treatment of choice for non-inflammatory acne.Benzoyl peroxide and/or topical antibiotics are used to treatinflammatory acne including papules pustules and cysts. Systemicantibiotics are also used for inflammatory acne.

Systemic therapy consists mainly of systemic antibiotics, usuallytetracycline, to decrease bacteria until the patient is in remission;then a lower dose is used for maintenance. Oral isoretinoin inhibitssebaceous gland function and keratinization by an unknown mechanism.However due to its severe side effects, including liver disease andbirth defects, its 16-20 week course is reserved for severe acneunresponsive to conventional therapy.

Seborrheic Dermatitis (Seborrhea)

Seborrhea is an acute or subacute skin disorder of unknown etiologypresenting as eruptions in skin areas containing many sebaceous glands.The scalp and face are most common and may result in hair loss(alopecia). Lesions are red to yellow and may be itchy and scaly.Treatment includes removal of scales with frequent washing andshampooing with selenium sulfide suspension, zinc pyrithione, or tar andsalicylate shampoo.

Sebaceous Cyst

Obstruction of a single sebaceous gland may result in an intra dermalcyst. These can occur anywhere on the body and become infected and formabscesses. Treatment includes oral antibiotics, surgical drainage and/orexcision of the cyst.

Seborrheic Blepharitis (Blepharitis)

The holocrine glands of the eyelid are called mebomium glands. Theyproduce an oily substance that aids in lubricating the exposed surfaceof the eye. Blepharitis is an acute to chronic condition that presentsas a burning and itching of the eyelids. Signs are waxy scales on theeyelashes, loss of eyelashes, and lid ulceration and secondary infectionwith Staphylococcus aureus.

Treatment includes meticulous hygiene, mild shampoo, and topicalantibiotics.

Rosacea and Rhinophyma

Although the cause of rosacea is unknown, it is closely associated withand involves sebaceous glands. Rosacea is a chronic condition thatbegins as periodic facial flushing and progresses to telangestasia,papules, pustules and nodules. It is more severe in men and oftenassociated with rhinophyma, thickened bulbous skin of the nose.Treatment of acne like rosacea includes topical or systemic antibiotics,topical steroids and Sulfacet-R lotion.

Furuncles, Carbuncles, Pustules, Chalazions, and Styes

Skin infections often begin in pilosebaceous glands. In acne theinfectious bacterium is P. acnes. However many conditions begin with aninflamed pilosebaceous gland and are secondarily infected with otherbacteria such as Staphylococcus aureus and Streptococcus epidermis.Single small infections are called furuncles, larger ones are calledpustules and when subdermally spread to create large fluctuant abscessescalled carbuncles. In the eye, analogous infections of the specializedholocrine glands are called styes and chalazions. Treatment of theseconditions includes warm compresses, topical and systemic antibiotics,and often surgical drainage.

Excessive Sebum

More of a cosmetic condition then a medical one, excessive sebumproduction is quite common. Most often the central area of the face isaffected, and this area looks and feels greasy. Treatment is frequentwashing of the face with strong soaps. This often causes secondarydrying of the remaining areas of the skin.

Excessive Cerumen

Cerumen is produced by mixed holocrine like glands in the skin of theear canal and its production is at least partly under cholinergiccontrol. Cerumen normally slowly migrates outward and is lost from themeatus of the ear canal. In some patients cerumen accumulates within theear canal, sometimes to the point of impaction. This can causeunderlying infection of the ear canal called otitis externa anddecreased hearing due to poor sound transmission. Treatment includescerumen dissolving chemicals such as carbamide peroxide and/or manualremoval of the wax by a physician.

Mammary Secretion

During and after pregnancy the mammary gland produces breast milk.Although lactation is principally hormonal the secretion and expulsionis influenced by neurotransmitters. Cattle with low grade botulinumtoxin poisoning have been noted to have dramatically decreased milkproduction despite normal appetites.

Lactation is natural and necessary for breast feeding the newborn.However not all mothers wish to breast feed, and in cases of miscarriageor stillborn the presence of breast fluids is a painful psychologicalreminder of the loss. Finally the increase in size of the breast duringthe pregnancy and post partum period eventually involutes, contributingto a cosmetic undesirable loss of tone in breast tissue.

U.S. 20020094339A1: Methods For Treating Mammary Gland Disordersdiscloses the use of CT to decrease the size of mammary glands andsecondarily decrease the incidence of mammary malignancies.

At present there is a large need in the art for compositions and methodsof inhibiting secretions of holocrine glands.

OBJECTS AND SUMMARY OF THE INVENTION

The present invention is directed to methods of controlling excessivelevels of secretions from glands selected from the group consisting ofholocrine glands, cerumen glands and mammary glands in patients byadministering to said patient a secretorily controlling amount ofbotulinum toxin. Such administration is particularly effective in thethinning the skin as well as smoothing of fine (as opposed to gross)wrinkles in the skin. It is also directed to the formulation ofcompositions for specifically carrying out such secretion control.

The methods disclosed are also directed to controlling secretions fromglands selected from the group consisting of holocrine glands, cerumenglands and mammary glands in patients whose level of glandular secretionis greater than is desirable by administering to said patient asecretorily controlling amount of botulinum toxin.

Administration may be by methodologies generally known to those skilledin the art such as topical and by injection, suitably subdermal,intradermal, transdermal and intramuscular injection. It should be notedthat the terms “dermal” and “cutaneous” are considered mutuallyequivalent.

Suitably, injection of botulinum toxin A is carried out at multiplesites in the skin, wherein the sites of adjacent injections areseparated by about 0.1 to 10 cm., suitably about 0.5 to about 5 cm.preferably by about 1.5 to about 3 cm. The toxins may be any of thebotulinum toxins A, B, C, D, E, F or G. The amounts administered mayvary between 0.1 and 1000 U, suitably about 1 to about 40, often fromabout 5 to about 10 U, depending on the manufactures specifications, theclass of the toxin and the mode of administration. Thus 1 U of Botoxequals about 2-4 units of Dysport and about 20-40 units of Myobloc.

The separation of the distances between injections will vary from about1 mm to about 10 cm, suitably from about 5 mm to about 5 cm, and moreusually from about 1 cm to about 3 cm. Thus for example botulinum A maybe suitably administered by intradermal injection between about 0.1 toabout 10 U at a separation of from about 0.5 to about 10 cm. preferablyat about 2.5 cm. Botulinum B may be administered in the range of 1-500U, preferably 100 U separated by 1.5 cm.

The repeat time range for these injections for maintenance of thedesired change varies substantially according to the location of theinjection, the condition to be adjusted and the condition of thepatient. Thus the repeat time may vary from about 1 week to about 50weeks, a common range is about 4 to about 25 weeks, or even about 12weeks to about 16 weeks.

These numbers are to be considered for exemplification and notlimitation Administration within any of the foregoing methods, amountsand separations may be established by one skilled in the art withoutundue experimentation for particular circumstances.

It is an object of the invention to provide compositions and methods forthe use of CT as treatment for clinical and cosmetic disorders of theskin.

It is another object of this invention to treat dysfunction of holocrineglands and related secretory structures in human skin with localapplications or injections of therapeutically effective amounts of CT orsimilar biologic agents to decrease the number and/or activity of axonsthat either directly or indirectly modulate the activity of theseglands.

It is an object of this invention to provide a treatment of skindisorders related to secretions of holocrine glands and relatedstructures, examples given without limitation the following: to decreaseproduction of secretion by blocking neural excitation of the secretorycells, the contractile components that express the secretion fromglands, the neural effects on glandular size, related skin cell changes,and the supply of fluid and nutrients by neural excitation. Holocrineglands include sebaceous glands, pilosebaceous glands, meibomium glands,glands of Zeiss and Moll, and the holocrine-like components of cerumenand mammary glands.

Conditions of excessive sebum production include acne vulgaris,seborrheic dermatitis, rosacea, rhinophyma, seborrheic blepharitis,sebaceous cysts, excess cerumen, and unwanted milk production. A secondbeneficial effect is to decrease bacterial infections of these glands bydecreasing the amount of secretion available for infection. Infectiousconditions include hidradenitis, furuncles, carbuncles, styes andchalazions. A third beneficial effect is to decrease gland size andproduction and related skin cell reactions for cosmetic benefit.Cosmetic conditions include mammary hypertrophy and to smooth skin anddecrease the size of skin surface pores.

DETAILED DESCRIPTION OF THE INVENTION

Sebum production and related disorders such as acne have long beenthought to be under the control of systemic hormones. Unexpectedly ithas been found that intradermal injection of CT decreases sebumproduction. The ability to decrease sebum production allows for thetreatment of a variety of new conditions that were not previouslytreatable with CT.

Moreover, without wishing to be bound by this theory, the mechanism bywhich CT decrease sebum production is by decreasing the release of theneuropeptide substance P.

By “Clostridia neurotoxins (CT)” it is meant the botulinum neurotoxinserotypes A-G produced by Clostridia botulinum, beratii, and butyricumand tetanus toxin produced by Clostridia tetani and other natural toxinswith similar biologic effects, particularly the proteolytic effects onSNARE proteins. The scope of the invention is meant to includemodifications where the modified CT or fragment thereof retainsessentially similar biological action as the wild type CT. Modificationsinclude, without limitation:

Hybrid CT that combine heavy and light chains, or fragments of thosechains, from different BT serotypes and TT.

Substitution of CT binding domain with, or the addition to the CT ofbinding domains from other bacterial toxins or viruses, ligands for cellmembrane receptors, antibodies or antibody fragments, combine fragmentsof different CT either with each other or with binding and/ortranslocation domains of other bacterial toxins.

Substitution of the CT translocation domain, or addition to the CT oftranslocation sequences from other bacterial toxins, or chemicallyconstructed translocation domains, or membrane transfer proteins such asthe TAT sequence.

Addition, substitution or subtraction of amino acids or chemicalmodification of amino acid side chains.

RECOMBINANT FORMS OF CT Delivery Within the Skin Cells of Nucleic AcidCoding for CT.

By “therapeutically effective amount” it is meant of purposes of thisinvention that the CT is administered in a non-toxic amount sufficientto cause reduction in the occurrence or magnitude of the symptoms beingtargeted. At present CT is measured by biological assay; a unit of BT isthe amount that causes death to 50% of mice when injectedintraperitoneally. BT A is marketed as Botox by Allergan Corp, IrvineCalif., and as Dysport by Ipsen Ltd, Berks United Kingdom. BT B ismarketed as Myobloc by Elan Pharmaceuticals, Dublin, Ireland. Other BTserotypes are available from Metabiologics, Madison, Wis. TT is notmarketed for human use but is from List Biological Labs(www.listlab.com).

A therapeutically effective amount of CT will vary depending on: thearea of skin to be treated as injections diffuse about 2 cm; the methodof application; the serotype of toxin used (approximately 20-40 BT-Bunits have the same effects as one BT-A unit in human clinicalapplications); the exact preparation of the serotype (potency of BT-Afrom the two commercial vendors varies. The same clinical effectrequires 2-4 times the dose of Dysport (from Ipsen) than Botox, the BT-Aformulation, from Allergan). At present the use of BT-A for muscleindications varies from about 1 unit (eye and larynx muscles) tohundreds of units (limb and major neck muscles). The exact dosage willnot require undo experimentation by those skilled in the art. (SaltzmanM: Drug Delivery, (2001) Oxford University Press, New York, N.Y.,Transdermal Drug Delivery Guy RH (editor) Marcel Dekker; (2003)).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A detailed method of CT application to reduce sebum production will bedisclosed and examples will be given to disclose minor variations fordifferent specific conditions.

EXAMPLE 1 Sebum Secretion Assay

Sebum secretion was assayed by application of fine blotting paper toforehead skin. The skin area was first wiped with moist and dry clothsto remove any skin secretions. Blotting at this time was negative. Twosheets of blotting paper were placed on both sides of forehead and anelastic band was then placed around the forehead to gently press theblotting paper onto the skin. After 2 hours the blotting papers wereremoved, allowed to dry and then transilluminated. Areas where sebumsecretion occurred were significantly darker then the unstained areas.The pattern of non-injected forehead skin was the of a circular areas ofapproximately 0.25 to 0.5 cm in diameter that overlapped extensivelyresulting in an almost complete dark band 4 cm wide across the forehead.

A 49 year old male was injected with 100 units of botulinum toxin B(Myobloc, Elan, Dublin). The injection was made with a size 26 needleattached to a 1 cc syringe. The skin of the right forehead was cleanedwith alcohol and the needle injection was placed into the center of theright forehead, equidistant from the eyebrow and hairline. The tip ofthe needle was advanced through the skin surface and injection was madeintradermally. A white raised bleb approximately 0.5 cm in diameter wasseen at the site of injection.

Directly after toxin injection, no change in blotting pattern was seenfrom uninjected patterns. One day after injection a repeat assay showedthat a clear area was present at the site of injection with a diameterof approximately 1 cm. On the second day after injection the area hadincreased to a maximum of 1.5 cm. The cleared area gradually decreasedin size and was not detectable at 2 months. In all trials the uninjectedside did not show any clear areas.

In another experiment on the same subject, 10 units of Botulinum A(Botox) at a dilution of 2.5 units per 0.1 cc of saline was injectedintradermally into the mid forehead. After one day a clear area wasfound after blotting with a maximum diameter of approximately 2.5 cm.Blotting showed a decrease in area by 50% by 2 months and was notdetectable at 4 months.

One surprising observation was that the injected area became noticeablysmoother within a few days after injection. This smooth appearance wasdue to smaller and less noticeable skin pores as well as thinning of theskin, and a decrease in the fine wrinkling of the skin surface. Both theflattening and smoothing effects lasted as long as the deceased sebumsecretion and the skin texture returned to its former condition afterthe other effects also disappeared. This smoothing is distinct from thedecrease in the larger skin wrinkles that occurs after facial skin isimmobilized by botulinum toxin injection into facial muscles ofexpression. The etiology of these effects on skin texture are unknown.However, without wanting to be bound by theory it may be due to atrophyof skin glandular elements and associated tissue due to denervation bythe botulinum toxin.

EXAMPLE 2 Acne Vulgaris

The target glands are sebum glands of the face, shoulders and back. BTwould be topically applied or injected so as to bind to the neuronsstimulating the pilosebaceous glands and associated structures, therebydecreasing the production and/or accumulation of sebum. This could bedone as treatment for exacerbations of the disease or as a preventivemeasure during quiescent periods.

A patient with a long history of acne vulgaris presents with multiplecomedones of both cheeks. Five injections of 2 units of BT-A areinjected intradermally into separate sites of each cheek for a total of20 units.

EXAMPLE 3

Seborrheic Dermatitis

A patient with seborrheic dermatitis presents with a widespread rash onher face and scalp associated with alopecia. Twenty injections of 2units of BT-A (total of 40 units) in injected into multiple sitesinvolved in the disorder including the area of hair loss.

EXAMPLE 4 Sebaceous Cyst

A patient presents with a 1 centimeter mildly tender sebaceous cystwithin the skin of the right inner thigh. A single injection of 5 unitsof BT-A is made into the cyst.

EXAMPLE 5 Rosacea and Rhinophyma

A patient with chronic rosacea and rhinophyma presents for treatment.Ten intradermal injections each containing 2 units of BT-A (total 20units) are made at various places of the mid face, and five additionalinjections are made directly into the skin of the nose.

EXAMPLE 6 Blepharitis

A patient with chronic blepharitis of the eyelids of both eyes. Thepatient receives a single injection of 0.5 units of BT-A to the edge ofeach eyelid (total 2 units).

EXAMPLE 7 Bacterial Infections

A patient with a history of recurrent eyelid chalzions applies topicalointment containing 10 units of BT-A per cc is daily along with warmcompresses.

EXAMPLE 8 Skin Texture Smoothing

A patient has significant fine wrinkling of her facial skin that shefeels is cosmetically objectionable. She undergoes topical applicationof 50 units of BT-A in a cream form to the skin of her forehead,periorbital region and cheeks.

The forgoing suppression of fine wrinkling is also by achieved by theintradermal injection of BT suitably at multiple but adjacent sites bythe injection of between 1 and 100 units of BT at separations of between0.5 to 10 cm. BT-A is preferred but BT-B (as well as BT C-BT G) may alsobe used.

Repetition of treatment at intervals of about 3 to about 6 months isdesirable.

EXAMPLE 9 Excessive Sebum

A patient has excessive sebum of the face requiring twice dailycleansing with special soaps that dry her skin. 10 injections, each of100 units of BT-B are injected into the skin of the eyebrows, forehead,and nose and nasolabial folds.

EXAMPLE 10 Unwanted Lactation

A female patient has post partum depression following a stillborn birth.To decrease the production of breast milk 3 injections of 20 units ofBT-A are made into each breast (120 units total).

EXAMPLE 11 Mammary Hypertrophy

A female patient with perceived mammary hypertrophy learns she ispregnant. Not wishing further enlargement of the breast during pregnancyshe receives 3 injections of 20 units of BT-A are made into each breast(120 units total).

EXAMPLE 12 Skin Cosmesis

A patient perceives that the skin of her nose has large pores related topilosebaceous glands. She is injected with two injections of 5 units ofBT-A (10 units total) to the skin overlying the nasal alae. In one monththe size of the skin pores has decreased by 50%.

EXAMPLE 13 Cerumen Blocking

A male patient experiences cerumen impaction every 6 months. Each ear isinjected with 5 units of BT-A into the skin at the junction of the bonyand cartilaginous canals.

REFERENCES

Abstracts of the International Conference 2002 Basic and TherapeuticAspects of Botulinum and Tetanus Toxins, Hannover, Germany, Jun. 8-12,2002

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W003/011333A1 botulinum toxin in the treatment or prevention of acne.

1. A method of treating skin cosmesis by decreasing skin pore size in asubject in need of such treatment, comprising administering to thedermis of said subject an effective amount of Botulinum toxin todecrease skin pore size in the subject.
 2. The method of claim 1,wherein the skin pores are large pores on the subject.
 3. The method ofclaim 2, wherein the large pores are decreased in size by at least 50%.4. The method of claim 1, wherein the subject's skin texture becomessmoother.
 5. The method of claim 1, wherein sebum production isdecreased in the subject, and the subject's skin is less greasy.
 6. Themethod of claim 1, wherein the administration is topical.
 7. The methodof claim 6, wherein the Botulinum toxin is administered transdermally.8. The method of claim 1, wherein the Botulinum toxin is Botulinum toxinA.
 9. The method of claim 1, wherein the administration is by subdermalor intradermal injection.
 10. The method of claim 9, comprisinginjection at multiple sites, wherein the sites of adjacent injectionsare separated by about 0.5 to 10 cm.
 11. The method of claim 10, whereinthe sites of adjacent injections are separated by about 1.5 to about 3cm.
 12. The method of claim 9, wherein the amount injected is between0.1 and 1000 U of Botulinum toxin.
 13. The method of claim 9, whereinthe amount injected is between 1 and 100 U of Botulinum toxin.
 14. Themethod of claim 9, wherein the amount injected is between 2 and 3 U ofBotulinum toxin.
 15. The method of claim 1, wherein said method isrepeated at intervals of from about 3 to about 6 months to inhibit saidrecurrence.
 16. The method of claim 1, wherein said method is repeatedat intervals of about 4 to inhibit said recurrence.
 17. The method ofclaim 1, wherein the Botulinum toxin is chosen from the group consistingof Botulinum toxin B, Botulinum toxin C, Botulinum toxin D, Botulinumtoxin E, Botulinum toxin F and Botulinum toxin G.